Yazarlar teknik destek ve yardım deneyler için Bayan Risa Otsuka teşekkür ederim. Bu araştırma kısmen Eğitim Bakanlığı, Bilim, Spor ve Kültür, tarafından desteklenmiştir Grant-Aid Genç Bilim Adamları (B), 2010, 22.700.476, ve KT Suzuken Memorial Foundation 2010 Ve bu çalışmanın Araştırma ve Geliştirme tarafından desteklenen için Yaşayan Madde, MEXT ve Nesil Süper Bilgisayar Projesi Geliştirme ve Kullanım bir parçası ve JST ERATO Suematsu Gaz Biyoloji Projesi tarafından kısmen ve Nesil Entegre Simülasyon.

1. Floresein izotiyosiyanat izomer I (FITC) ile Eritrositler Etiketleme Kullanılan tüm deneysel protokolleri Keio Üniversitesi Tıp Fakültesi Hayvan Bakım Komitesi tarafından onaylandı. <ol><li> Bir donör fareler uyutmak ve yaptıktan sonra bir Seliotomide kesi heparin az miktarda içeren 1 ml şırınga ile vena kava tam kan çekilme. Fare hemen sodyum pentobarbital enjeksiyon (100 mg / kg ip) tarafından ötenazi edildi. </li><li> Bir sallanma-kova rotor içinde 400 g hızında 5 dakika süreyle santrifüjlenerek PBS (pH 7.4) ile iki kez tam kan yıkanır. </li><li> 100 mM Na 2 HPO 4, 5 ml içinde FITC 10 mg eritilir ve 0.22 um-gözeneklere sahip olan bir zar ile filtre. </li><li> Bir test tüpü 3 mM glukoz 0.15 ml, 180 mM 100 mM NaH 2 PO 4 ve 1.5 ml 2 HPO 4 Na 0.25 ml FITC çözeltinin 1 ml karıştırın. R 0.2 ml ilave edilirM.Ö. pelet ve karıştırma için tüp dokunun. </li><li> 4 ° C&#39;de 2 saat boyunca boru tutmak ve sonra iki defa PBS ile boyanmış kırmızı kan yıkanır. </li><li> Slayt çim eritrosit süspansiyonu küçük bir miktar koyun ve eritrositler sağlıklı bakarsanız bakın (yani, onların şekil ve boyutları normal olduğunu) ve floresan yeterli olduğunu. </li><li> PH 7.4 &#39;te PBS 0.9 ml RBCs 0.1 ml Askıya, ve 4 süspansiyon tutmak ° C enjeksiyona kadar. </li></ol> 2. Oksijen-duyarlı Dye hazırlanması <ol><li> PBS pH 7.4 &#39;te 10 ml BSA, 500 mg eritilir. </li><li> Pd 30 mg ekleme (II)-mezo-tetra (4-karboksifenil) BSA çözeltisi ile porphine (Pd-TCPP) ve gece boyunca karıştırılır. </li><li> (İsteğe bağlı) ücretsiz Pd-TCPP ayırmak için yerçekimi akışı kromatografisi veya bir spin kolon kullanarak BSA bağlı Pd-TCPP ayıklayın. </li><li> Pd-TCPP çözünmemiş kaldırmak ve 0.22-mikron gözeneklere sahip bir membran filtre ile süpernatant filtrelemek için çözüm santrifüjleyin. &lt;/ Li&gt; <li> -20 ° C&#39;de tüpler Mağaza 1 ml hacimde Tekrarlanan donma-erime döngüleri kaçının. </li></ol> 3. Hayvan Hazırlık <ol><li> Mikrodolaşım mikroskobik gözlem için delik üzerinde 30 mm kare kare cam kapak bir delik 20 mm çapında ve bant ile plastik bir tabak hazırlayın. </li><li> Bir fare uyuştur, deri kürk kaldırmak ve hazırlık. Heparinize PBS ile doldurulmuş bir 10 cm&#39;lik bir polietilen (PE 10) bağlı bir kateter 30 G iğne kullanılarak madde enjeksiyon için boşa kuyruk bir kateter yerleştirin. </li><li> Median insizyon sonra, plastik plaka üzerinde bir karaciğer ana lobülü genişletmek ve sternal pozisyonda bulunan fare yerleştirin. </li><li> Solunum ile karaciğerin hareketli ve kurumasını tutarak inhibe hepatik lobun kenarında mutfak wrap (3 mm x 8 mm) ve çini onlarla küçük fişleri hazırlayın. </li><li> Mikroskop altında hepatik mikrosirkülasyonu (iletilen ışık ile) dikkat edin, onaylayın ki blood akışı, en azından 15 dakika süreyle görüş alanını herhangi bir durağanlığın sahiptir. </li><li> Yavaş yavaş kan akımı gözlem veya pO 2 ölçümü için Pd-TCPP çözüm 0.2 ml floresan ile işaretlenmiş eritrositler 0.2 ml enjekte edilir. FITC-etiketli eritrositler Bu miktar görüntülenmiştir bölgede dolaşımda bulunan tüm eritrositler 1/50 sorumlu olacaktır. </li></ol> 4. Kan Akış Görselleştirme <ol><li> Bandpass (450-490 nm) 1 geçti civa lambası ile irradyasyonu ile FITC heyecanlandırıyor. </li><li> Bir CCD kamera ile floresan görüntüyü kaydedin. </li></ol> 5.. pO 2 Ölçme Pd-TCPP fosforesans oldukça zayıf olup, yüksek hassasiyetli dedektör ile tespit edilmelidir. Bütün deneyler bir karanlık odaya yapılması gerekir. <ol><li> Pd-TCPP emilimi pikleri 410 nm ve 532 nm, bu nedenle ikinci harmonik dalga boyu 532 nm eksitasyon için tavsiye edilir. YAG lazer pulse 2: Bu dalga boyu Nd tarafından oluşturulabilir. </li><li> Inverted mikroskop uygun portuna YAG lazer ve odak düzlemi merkezi bir konuma ışın spot ayarlayın: Nd ışın besleyin. Uzaysal çözünürlüğü, bir iğne deliğinden ışını geçirilerek değiştirildiğinde lazer spot büyüklüğü, bağlıdır. </li><li> Fosforesans saptamak için, uzun bir geçiş filtresi (&gt; 620 nm) ve mikroskop diğer portuna bir photomultiplier tüpü (PMT) takabilirsiniz. PMT özellikle 700 nm çapında, kırmızı dalga boyları karşı duyarlı olmalıdır. </li><li> Fosforesans sinyali (500 puan 200 KHz) tadın ve çürüme bir üstel fonksiyonu takarak çürüme zaman sabitini hesaplayabilirsiniz. </li><li> Kalibrasyon için pO 2 150 mmHg ve 0 mmHg Pd-TCPP çözüm örneklerinin iki set hazırlayın. Oksijen yokluğunda üretmek için örneklemek üzere sodyum ditionit hacim olarak% 1 ekleyin. 7,4 azından örnekleri arasında pH ve sıcaklık tutmakKalibrasyon sırasında 37 ° C &#39;de Ture. </li><li> Stern-Volmer sabiti k q ve oksijensiz lüminesans yıkım zamanı denklemde τ 0 (1) 3 Fix. 37 ° C ve 7.4 pH Pd-TCPP çözümleri ile bizim durumda, k q 374 mmHg -1 s -1 ve τ 0 0.74 ms. Bu ölçüm parametreleri ekipmanı (yani lazer, dedektör, diğer optik cihazlar) ve sinyal işleme yöntemine bağlıdır, bu yüzden her kalibrasyon ölçüm sistemi 4 gereklidir. τ 0 / τ = ​​1 + q k 0 • • τ pO 2 (1) </li><li> Mikroskop sahnede plaka üzerinde fare ayarlayın, hepatik mikrosirkülasyonu gözlemlemek ve lazer nokta nokta hedef mikrodamar pozisyonunu ayarlayın. </li></ol> 6. Hepatit modeli <ol><li> Gecede suya ücretsiz erişim ile hızlı fare. </li><li> Bir hazırlayın20 DMSO asetaminofen (APAP) ml çözelti başına mg. </li><li> 1 ml/100 gr bw (yani, 200 mg / kg) intraperitoneal olarak enjekte. Enjeksiyonundan sonra, fare yiyecek ve su 5 serbest erişim verilebilir. </li><li> Enjeksiyondan 24 saat sonra, fare uyutmak ve median insizyon yaparak karaciğer maruz bırakmaktadır. Hepatit elde edilirse, perisentral bölgede nekroz çıplak gözle kolayca görülebilir. </li></ol> 7. Temsilcisi Sonuçlar Hepatik mikrodolaşım Örneği görüntü (a) iletilen bir ışıklı görüntü ve (b), flöresanlı bir resimdir Şekil 1 &#39;de gösterilmektedir. Bireysel FITC etiketli eritrosit video film gözlenir ve portal venlerde, sinüzoidler ve santral venlerde kan akış yönü ile tanınır. Şekil 2 de gösterildiği gibi, bir x100 objektif lens ile merkezi bir venül irradyasyonu laser ışınının approxim idiçapı 10 um rilmesi gerekir. Hepatik mikrosirkülasyonda İntravasküler pO 2 erkek C57BL / 6 farelerde ölçülebilir ve Şekil 3 (a) pO 2 ve portal ve santral venlerde damar çapı arasındaki ilişkileri gösterir edildi. Sinüzoidler geçerken merkezi venüller portal gelen pO 2 degrade etkili olduğunu eritrositlerde serbest oksijen gösterir. Şekil 3 (b) &#39;de gösterildiği gibi, ortalama pO2 portal damarlar, sinüzoidlerdeki 48.2 mmHg ve santral venlerde 38.9 mmHg 59.8 mmHg idi. Biz APAP intraperitoneal enjekte edilerek akut hepatit üretilen zaman, intravasküler pO 2 önemli ölçüde hepatik mikrodolaşım her kesiminde artmıştı: p sinüzoidler ve santral venlerin (Şekil 3 (c)), portal ven ve p &lt;0.01 &lt;0.05. <img src="/files/ftp_upload/3996/3996fig1.jpg" alt="Şekil 1" /> Şekil 1. Düşük büyütme images (a) geçen ışık ve (b) flüoresan ile hepatik mikrodolaşım gösteren. <img src="/files/ftp_upload/3996/3996fig2.jpg" alt="Şekil 2" /> Şekil 2. PO 2 ölçüm için hedeflenen merkezi venül irradyasyonu lazer nokta görüntüsü. <img src="/files/ftp_upload/3996/3996fig3.jpg" alt="Şekil 3" /> Sağlıklı kontrol farelerin ve asetaminofen bağlı karaciğer hasarı ile farelerde karaciğer mikrosirkülasyonda Şekil 3. PO 2 degrade. (A) pO 2 karşılık portal venüller (FV) ve merkezi venüller (KV) için damar çapı gösterir, ve (b) sinüsoidlerinden (S) vasıtasıyla PV CV oksijen gradyanı gösterir. PV ve CV arasındaki pO 2 farkı hepatositler veya diğer parankimal hücrelerin oksijen tüketimi yansıtır. (C) APAP alımının yol açtığı hepatit durumunda, pO 2 önemli ölçüde PV, S ve CV ve pO 2 yükselebilir olduğunu gösterirdegrade hepatik oksijen tüketimi tatlıya belirten azalmıştır.

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Biz ifşa hiçbir şey yok.

Dokularına oksijen taşınmasına mikrodolaşımında önemli bir rolü olduğunu ve bir çok gazete mikrodamar anatomi ve reoloji 6 ile ilgili hastalıklar açıklar. Karaciğer kan akımı arteriyel ve venöz kan bir kombinasyonudur. Karaciğer kan akımının yaklaşık% 30 portal ven tarafından sağlanan hepatik arter ve diğer% 70, sağladığı iyi oksijenli kan ise, dalak ve gastrointestinal kanal 7 den az oksijenli kan venöz çıkış olduğunu. Bu tür yağlı karaciğer, şok, veya tü...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Pd (II) mezo-tetra (4-karboksifenil) porphine </td><td> Frontier Scientific, Inc </td><td> PdT790 </td></tr><tr><td> Floresein izotiyosiyanat izomeri I </td><td> Sigma-Aldrich Co </td><td> F7250 </td></tr><tr><td> Asetaminofen </td><td> Sigma-Aldrich Co </td><td> A7085 </td></tr></tbody></table> <table border="1"><tbody><tr><td> Ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td></td><td></td><td></td><td> Ekipman </td></tr><tr><td> photomultiplier tüp </td><td> Hamamatsu Photonics KK </td><td> H10722-20 </td></tr></tbody></table>

visualization and analysis of blood flow and oxygen consumption in hepatic microcirculation: application to an acute hepatitis model

Hepatik oksijen tüketimi nispeten yüksektir ancak hepatik kan akımı yaklaşık% 70 az gastrointestinal sistem ve dalak türetilen portal ven kan oksijenli olduğu için oksijen kaynağı ve karaciğerde talep arasındaki önemli bir fark vardır. Oksijen sinüzoidler üzerinden merkezi bir venül için portal ven bir terminal dalı akan kan ile hepatositler teslim ve bu hepatik lobülleri bir oksijen gradiyenti yapar. Oksijen gradyanı yukan ve aşağı hepatik mikrodolaşım Enzimlerin ifadesi, fakat hepatik mikrodolaşım oksijen tüketimi ölçülmesi için teknikler eksikliği karaciğer içindeki oksijen metabolizması ilgili mekanizmaları aydınlatılması geciktirmiştir içerir önemli fiziksel bir parametredir. Bu nedenle karaciğer mikrosirkülasyonu görselleştirmek için FITC etiketli eritrositleri kullanılmış ve burada kılcal oksijen kısmi basıncı ölçmek için lazer yardımlı phosphorimetry kullanılır. Noncontact ve sürekli optik ölçüm kan akım hızları, damar çapları ve karaciğerde oksijen tüketimi ile ilgili oksijen degradeler ölçmek olabilir. Akut hepatit modeli biz perisentral bölgesinde hepatosit nekrozu oksijen basıncı yukarı kayar ve enzim ifade etkileyebilir belirten, biz portal ve santral venüller hem artmış oksijen basıncı ancak sinüzoidlerdeki azalmış oksijen gradiyenti gözlendi farelere asetaminofen ile sağlandı periportal bölgede. Sonuç olarak, ölçme hepatik hemodinamik ve oksijen tüketimi için optik yöntemlerle karaciğer hastalığı ile ilgili mekanizmaların ortaya çıkarabilir.

Watch this Scientific Journal Video about Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model at JoVE.com

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Bir optik sistem FITC-etiketli eritrosit ile karaciğer mikro görselleştirmek ve lazer yardımlı phosphorimetry ile kılcal oksijen kısmi basıncı ölçmek için geliştirilmiştir. Bu yöntem, mikrovasküler yapısı, çapı, kan akış hızı, ve oksijen gerilimi analiz ederek fizyolojik ve patolojik mekanizmalar araştırılması için de kullanılabilir.

Akut Hepatit Model Uygulama: Görselleştirme ve Hepatik Mikrosirkülasyon Kan Akış ve Oksijen Tüketimi Analizi

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

visualization-analysis-blood-flow-oxygen-consumption-hepatic

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JoVE Journal

Medicine

12.5K Görüntüleme.  Keio University. Bir optik sistem FITC-etiketli eritrosit ile karaciğer mikro görselleştirmek ve lazer yardımlı phosphorimetry ile kılcal oksijen kısmi basıncı ölçmek için geliştirilmiştir. Bu yöntem, mikrovasküler yapısı, çapı, kan akış hızı, ve oksijen gerilimi analiz ederek fizyolojik ve patolojik mekanizmalar araştırılması için de kullanılabilir.

Video: Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Murin Bioluminescent Karaciğer Tümör Modeli

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Tıp

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

Heterotopik Yardımcı Sıçan Karaciğer Nakli ile Akut Karaciğer Yetmezliği Portal Ven arteriali Akış ayarlı

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Subaraknoid Kanama Akut ve Geç sekeli Çalışmaları Tavşan Kan şant Modeli: Teknik Yönleri

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

Deneysel Spinal Kord Yaralanmalarında Omurilik Kan Akımının Değerlendirilmesi için geliştirilmiş Ultrason Görüntüleme Kontrast

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Bir oksijen-Glukoz Yoksunluk ve reoksijenizasyonun<em&gt; In Vitro</em&gt; İskemi-Reperfüzyon Modeli incelenmesi Kan-Beyin Bariyeri Disfonksiyon için

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

Tedavi Kan Retina Bariyer ihlali ve Potansiyel İlaçlar değerlendirilmesi için bir Akut Retina Modeli

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Optimize Analizi<em&gt; İn Vivo</em&gt; ve<em&gt; İn Vitro</em&gt; Karaciğer Steatoz

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Bir Ameroid yılanı kullanarak domuz kronik kalp iskemi modelinde

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Oksijen tüketim oranını bir hücre dışı akı Analyzer kullanarak birincil kültürlü fare yenidoğan Cardiomyocytes içinde analiz

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.